SS is a neuropeptide which can act as a hormone, a neurotransmitter or an autocrine regulatory factor to inhibit secretory processes in a variety of endocrine, exocrine and neuronal target cells. Dr. Schonbrunn has shown that SS acts to inhibit pituitary hormone secretion via a plasma membrane receptor which is coupled to one or more pertussis toxin sensitive G proteins. SS receptors activate multiple transduction pathways, including inhibition of adenylylate cyclase, stimulation of potassium conductance and inhibition of calcium conductance. However, the structures of SS receptors are unknown. Indeed, it is not clear whether subtypes of SS receptors exist and, if so, whether they activate the same or different membrane effectors. To address these problems, the investigator propose to clone cDNAs for SS receptors and examine their function individually in mammalian expression systems. Initial studies will focus on cloning the cDNA encoding the 85,000 dalton protein which she has recently identified as a SS receptor by photoaffinity labelling. The general strategy to be employed will involve purifying the 85 kDa receptor protein, obtaining amino acid sequences for peptide fragments from the receptor, and using these sequences to synthesize appropriate probes for screening a cDNA library. Verification of the identity of the putative SS receptor clones will be accomplished (a) by sequencing the clones and comparing the deduced amino acid sequence from the cDNA to the peptide sequences obtained from the purified receptor, and (b) by expressing the cDNAs in receptor negative mammalian cell lines containing the required transduction machinery and demonstrating the functional capabilities of the resulting protein. Having determined the structure of this 85 kDa SS receptor from the cDNA sequence, she will identify related cDNAs by homology cloning under low stringency conditions. Expression assays will again determine if these cDNAs code for functional SS receptor subtypes with different analog specificities or transduction mechanisms. If so, these cDNA's will be sequenced to elucidate the structures of SS receptor subtypes and used to determine the distribution of mRNAs coding for the individual members of this receptor family. To enable her to measure SS receptor proteins, she will generate antibodies to SS receptors by using appropriate expression systems to produce large amounts of each receptor and receptor fragments for immunization. The antibodies and the cDNA's will be used to quantitate the level of SS receptor protein and mRNA following treatment of pituitary cells with agents previously shown to affect receptor density by binding measurements. In addition, she will determine whether receptor phosphorylation is involved in the regulation of SS receptor function. Together, these studies will elucidate for the first time the structure, function and regulation of SS receptors at a molecular level.